Nature - USA (2020-10-15)

Article

RmCdnE N166

CgCdnE D227

FsCdnE D233

cGAS S378

DncV S260

U

CdnE homologue

s

STING-associated

Flavobacteriaceae sp.

Capnocytophaga granulosa

Aggregatibacter actinomycetemcomitans

Bacteroides fragilis

Sphingobacterium faecium

Flavobacterium daejeonense

Lachnospiraceae bacterium

Niabella drilacis

Pedobacter luteus

Riemerella anatipestifer

Chitinophaga polysaccharea

Hymenobacter sp.

Pseudomonas aeruginosa

Escherichia coli

Elizabethkingia meningoseptica

Staphylococcus aureus

Enterococcus faecalis

Legionella pneumophila

Vibrio cholerae DncV

Homo sapiens cGAS

D233

analogous residue

D D D D D D D D D D D D N N N N N S S S

c-di-GMP

c-UMP–AMP

3',3'-cGAMP

3',3'-cGAMP

2',3'-cGAMP

CDN

produced

d

e

Ori.

Pi

c-di-GMP

[α^32 P] NTPs:

NTPs:

WT

N

N

A

N

C

N

G

N

U

N

G

G

G

G

FsCdnE Mutant: D233N

N

N

A

N

C

N

G

N

U

N

G

G

D233S

N

N

A

N

C

N

G

N

U

N

G

G

D233A WT

G

G

Ori.

Pi

c-di-GMP

FsCdnE Mutant: WTD233

N

D233

S

D233

A

1 min 5 min 20 min

WTD233

N

D233

S

D233

A

WTD233

N

D233

S

D233

A

Time:

f

a c

Ori.

Pi

c-di-GMP

[α^32 P] NTPs:

NTPs:

WspR

N

N

A

N

C

N

G

N

U

N

G

G

G

G

ReCdnE (TM)

N

N

A

N

C

N

G

N

U

N

G

G

CgCdnE (TIR)

b

WspR

N

N

A

N

C

N

G

N

U

N

G

G

G

G

LbCdnE (TIR)

Ori.

Pi

c-di-GMP

Nuclease:

Reaction: DncVcGASFsCdnE

P1

3',3'-cGAMP

2',3'-cGAMP

— P1 + CIP

DncVcGASFsCdnEDncVcGASFsCdnE

Observedm/z = 691.102

Expected [M+H]+m/z = 691.102

c-di-GMP

Chemical formula: C 20 H 25 N 10 O 14 P 2

OH

O

OHO

OPO

NH

N

N

O

N NH 2

O

O OH

PO

OH

O

HN

N

N

O

H 2 N N

Extended Data Fig. 3 | Biochemical analysis of c-di-GMP synthesis by bacterial
STING-associated CD-NTases. a, In addition to FsCdnE (Fig. 2b), CdnE
homologues from three divergent STING-containing CBASS operons were
purified and tested for cyclic-dinucleotide-synthesis specificity using α^32 P-
radiolabelled NTPs and thin-layer chromatography. Deconvolution experiments
show a single major product requiring only GTP that migrates identically to
c-di-GMP synthesized by the GGDEF enzyme WspR. All reactions were treated with
alkaline phosphatase to remove exposed phosphates. Only two bacterial genomes
encoding a STING-containing CBASS operon retain proteins with a predicted
canonical GGDEF c-di-GMP signalling domain. The exceptions are chlorobi
bacterium EBPR Bin 190, which contains a single GGDEF domain that is fused
to a SLATT domain and may be part of a CBASS-like system^10 , and a Lachnospiraceae
bacterium RUG226 genome that encodes many GGDEF genes. The Lachnospiraceae
bacterium RUG226 CdnE retains exclusive production of c-di-GMP suggesting the
CdnE–STING system is sequestered in this bacterium or that an unknown
mechanism may exist to prevent toxic STING activation. ReCdnE, Roseivirga
ehrenbergii; CgCdnE, Capnocytophaga granulosa; LbCdnE, Lachnospiraceae
bacterium; N, all four rNTPs; Pi, inorganic phosphate; ori., origin. Data are
representative of two independent experiments. b, Nuclease treatment confirms
that the FsCdnE enzymatic product contains only canonical 3′–5′ phosphodiester
bonds. The [α^32 P]GTP cyclic dinucleotide product is susceptible to cleavage by
nuclease P1 resulting in release of GMP as a new species, which migrates further up
the TLC plate. Further digestion with calf-intestinal phosphatase (CIP) removes all
exposed phosphates, resulting in complete loss of a labelled product spot. DncV
(Dinucleotide cyclase in Vibrio)-derived 3′,3′-cGAMP is similarly susceptible to

complete digestion by P1 and CIP treatment, whereas 2′,3′-cGAMP synthesized by

mouse cGAS is only partially digested owing to the presence of the non-canonical

2′–5′ bond. Data are representative of two independent experiments. c, High-

resolution mass spectrometry analysis confirms the identity of the major FsCdnE

enzymatic product as canonical c-di-GMP. Chemically synthesized c-di-GMP was

used for direct spectral comparison. d, Sequence alignment and enlarged inset of

the active-site of the RmCdnE structure in complex with nonhydrolyzable UTP and

ATP analogues (PDB 6E0L), demonstrating a contact in the CD-NTase lid domain

known to control nucleobase sequence specificity^15. RmCdnE synthesizes cyclic

UMP–AMP and uses N166 to specifically contact the uridine Watson–Crick edge.

By contrast, FsCdnE and CgCdnE contain an aspartic acid substitution at this

position and synthesize c-di-GMP, and V. cholerae DncV and human cGAS contain a

serine substitution at this position and synthesize 3′,3′-cGAMP and 2′,3′-cGAMP. An

aspartic acid at the FsCdnE D233 position is conserved among 93% of STING-

associated CD-NTase enzymes (96 of 103), consistent with strict specificity of c-di-

GMP as the nucleotide second messenger that controls bacterial STING activation.

RmCdnE: PDB 6E0L^15 ; V. cholerae DncV: PDB 4TY0^57 ; and human cGAS: PDB 6CTA

(DNA omitted for clarity)^37. e, f, Mutational analysis of the importance of D233 in

FsCdnE c-di-GMP synthesis activity. D233 substitutions do not disrupt the overall

ability of FsCdnE to selectively synthesis c-di-GMP, but a D233A substitution

causes a mild reduction in nucleobase selectivity and efficiency of c-di-GMP

synthesis. These results are consistent with a role for D233 in nucleobase selection

but demonstrate full selectively is achieved by additional contacts in the active site

pocket. Data are representative of three independent experiments.